Wilm and Mann.sup.1-3 demonstrated the use of metal coated glass Electrospray (ES) tips with micron size orifices to study proteins and peptides. They Electrosprayed sample using small diameter tips and demonstrated that low nanoliter per minute liquid flow rates were possible with total sample consumption of less then one microliter. Less than one microliter of sample bearing liquid can be loaded into a glass tube with a sharply drawn exit end tip (microtip). The Electrospray flow rates with these tips can typically be 25 nanoliters per minute allowing analytical ES mass spectroscopy run times exceeding thirty minutes for one microliter of sample solution loaded. Extensive mass analysis including Collisional Induced Dissociation (CID) and MS/MS studies can be performed automatically or manually in this time frame with minimum sample consumption.
A glass replaceable Electrospray microtip can be fabricated by drawing a small glass tube out to a point which may have an inner diameter on the order of one to a few microns in diameter and a wall thickness at the tip of less than ten microns. This smaller inner diameter and small ES tip outer diameter results in a Taylor cone with and a reduced base diameter. The lower liquid flow rates result in a smaller filament diameter extending from the Taylor cone when compared to the filament diameters formed from higher flow rate Electrospray applications where larger bore ES tips are used. In Electrospray operation, charged droplets are formed by breaking off the liquid filament protruding from the tip of the stable Taylor cone. The Electrosprayed charged droplets produced from microtips are smaller in diameter than one micron and are not generally visible with forward light scattering and magnification as is the case with higher flow rate Electrospray operation. The lower liquid flow rates and smaller charged droplet sizes produced from Electrospray with microtips allow higher sampling efficiency and improved sensitivity when compared with higher flow rate Electrospray. Sensitivity is defined here as signal to noise achieved versus sample consumed. The reduced diameter of the ES microtips also allows the unassisted Electrospraying of aqueous solutions or aqueous solution containing acids or buffers. Electrospraying of such solutions is required for example when mass analyzing proteins in an active or native folding pattern. The increased sensitivity, longer working time and greatly reduced consumption of sample has led to increasing use of ES/MS operation with reduced diameter Electrospray tips, referred to herein as microtips.
Replaceable microtips have been fabricated with metal tubes, fused silica tubes and borosilcate tubes. Borosilcate tubes pulled down to a fine tip and gold coated on the tip outer surface have become the most common type of microtip used in the field. This combination is primarily due to the ease and low cost of uniformly fabricating such tips from standard glass tube sizes. Depending on the tip drawing and metal coating process used, microtips tips have been fabricated with closed and open ends and with widely varying quality in metal coating operational longevity. In practice, conductive paste is often used to insure electrical contact between the metal coated tip and the microtip holder. This is undesirable due to increased setup time and due to the addition of a contaminating substance which can produce unwanted mass spectral background peaks during Electrospray operation. In most commercially available Electrospray sources which include microtips, the microtips must be operated at hundreds or thousands of volts to achieve stable Electrospray. High voltage applied directly to the microtip instead of ground potential has made some Electrospray configurations less safe during operation due to exposed high voltage in some designs. In previous Electrospray source designs, high voltage applied to the microtip must be turned off to insure user safety when microtips are exchanged. Changing the ES source conditions or partially disassembling or opening the ES source to exchange microtips can be inconvenient and inefficient when trying to maximize sample throughput.
The invention includes a new Electrospray probe with removable microtips apparatus with improved methods of microtip setup and installation. The ES probe design facilitates the removal and installation of microtips. The design allows for automatic and reliable electrical contact at the Electrospray tip for metal coated or uncoated microtips without the need for conductive paste or other contaminants added to the tip. The ES probe accepts a range of microtip sizes and types. Axial and radial adjustment is included in the ES probe to allow optimal positioning of the microtip with the orifice into vacuum in the ES atmospheric pressure chamber. The invention includes a means of forming an electrical contact with the ES probe when using fused silica or borosilcate microtips having no conductive coating. For such microtips, one embodiment of the invention includes a conducting wire placed along the inside bore of the microtip which is held in place with an external O-ring. The O-ring is positioned in a manner which insures electrical contact between the wire and the ES probe when the microtip is installed. Microtips which are fabricated with and open tip orifice can be used without modification in the ES microprobe apparatus. Dipping such an open tipped microtip into a solution, particularly solutions with low surface tension, prior to loading the sample will aid in bringing the sample to the tip when initiating Electrospray. This method is particularly useful when Electrospraying high surface tension aqueous solutions through very small bore microtips. The inventions improve the reliability of Electrospray operation with microtips and increase the operational longevity of each microtip. A large range of coated and uncoated tip styles can be installed in the new Electrospray probe apparatus. The probe assembly includes the option of applying gas flow locally at the tip during ES operation. Such an option may be used to suppress corona discharge while Electrospraying aqueous solutions or running in negative ion mode. The new apparatus combined with improved microtip setup methods increases the ease of use and reduces the cost of running samples with microtip low flow rate Electrospray. One embodiment of the invention allows the safe and convenient exchange of microtips without the need to turn off the high voltage potentials in the Electrospray atmospheric pressure source chamber even if voltage other than ground potential is applied directly to the microtip itself during operation. This embodiment eliminates the cost and complexity of including safety voltage shutoffs for the Electrospray chamber and enables the user to rapidly and efficiently exchange Electrospray microtips with a minimum of down time.